The targeted inhibition of gene expression is an essential tool for basic medical research in numerous fields and has many potential clinical applications. However, improved tools are needed for probing the functions of cloned genes, especially in light of the comprehensive genome sequencing projects now underway for many organisms. The present proposal is directed at understanding and improving one such tool known as "RNAi" for RNA mediated genetic interference. RNAI was discovered in the course of attempts to use "antisense" methodology to interfere with gene expression in C. elegans. These studies led to the surprising observation that preparations of either "sense" or "antisense" RNA strands can induce potent and specific genetic interference upon microinjection into this organism. Findings described in this proposal suggest that a cellular and organismal response may underlie this genetic interference. Remarkably, interfering effects are observed not only throughout the tissues of the injected animal but are inherited by 100% of the injected animal's progeny. In subsequent generations genetic interference can be transmitted in the sperm or oocytes as a dominant extragenic factor. Microinjection of RNA (but not DNA) can induce interference. Surprisingly, double stranded RNA is much more effective at inducing interference than is either single strand. This proposal outlines a systematic approach that will dissect the molecular and genetic mechanisms that underlie RNAi. Aims will include: properties associated with interference. Improvements in the methodology. And identification of the C. elegans genes whose projects mediate genetic interference. Insights from these studies may lead directly to the development of related RNA interference technologies for probing gene function in other organisms including humans.